Micrococcus luteus teichuronic acids activate human and murine monocytic cells in a CD14- and toll-like receptor 4-dependent manner

Abstract

Teichuronic acid (TUA), a component of the cell walls of the gram-positive organism Micrococcus luteus (formerly Micrococcus lysodeikticus), induced inflammatory cytokines in C3H/HeN mice but not in lipopolysaccharide (LPS)-resistant C3H/HeJ mice that have a defect in the Toll-like receptor 4 (TLR4) gene, both in vivo and in vitro, similarly to LPS (T. Monodane, Y. Kawabata, S. Yang, S. Hase, and H. Takada, J. Med. Microbiol. 50:4-12, 2001). In this study, we found that purified TUA (p-TUA) induced tumor necrosis factor alpha (TNF-alpha) in murine monocytic J774.1 cells but not in mutant LR-9 cells expressing membrane CD14 at a lower level than the parent J774.1 cells. The TNF-alpha-inducing activity of p-TUA in J774.1 cells was completely inhibited by anti-mouse CD14 monoclonal antibody (MAb). p-TUA also induced interleukin-8 (IL-8) in human monocytic THP-1 cells differentiated to macrophage-like cells expressing CD14. Anti-human CD14 MAb, anti-human TLR4 MAb, and synthetic lipid A precursor IV(A), an LPS antagonist, almost completely inhibited the IL-8-inducing ability of p-TUA, as well as LPS, in the differentiated THP-1 cells. Reduced p-TUA did not exhibit any activities in J774.1 or THP-1 cells. These findings strongly suggested that M. luteus TUA activates murine and human monocytic cells in a CD14- and TLR4-dependent manner, similar to LPS.

FIG. 1

Chemical structures of p-TUA and p-TUA-[H], based on previous reports (14, 26).

FIG. 2

mCD14 expression in J774.1 and LR-9 cells in culture. J774.1 (A) and LR-9 (B) cells were stained with fluorescein isothiocyanate-labeled anti-mouse CD14 MAb (rmC5-3) and analyzed by fluorescence-activated cell sorter analysis. The results shown are representative of those from three different experiments.

FIG. 3

Induction of TNF-α by p-TUA in J774.1 and LR-9 cells. J774.1 (A) and LR-9 (B) cells were stimulated with p-TUA and reference Salmonella serovar Abortus-equi LPS at the indicated concentrations for 24 h in triplicate. The TNF-α levels in the culture supernatants were determinated by ELISA, and are expressed as means ± standard deviations. ∗, P < 0.05; ∗∗, P < 0.01 (versus medium alone). The results are representative of those from three different experiments.

FIG. 4

Induction of TNF-α by PMA in J774.1 and LR-9 cells. J774.1 (A) and LR-9 (B) cells were stimulated with phorbol myristate acetate (PMA) and reference Salmonella serovar Abortus-equi LPS at the indicated concentrations for 24 h in triplicate. The TNF-α levels in the culture supernatants were determinated by ELISA and are expressed as means ± standard deviations. ∗∗, P < 0.01 versus medium alone. The results are representative of those from two different experiments.

FIG. 5

Blocking effect of anti-mouse CD14 MAb on TNF-α secretion by J774.1 cells in response to p-TUA. J774.1 cells were preincubated with anti-mouse CD14 MAb 4C1 or isotype-matched control antibody (50 μg/ml) for 30 min and then stimulated with p- TUA (50 μg/ml) and reference LPS (10 ng/ml) for 24 h. The levels of TNF-α in the culture supernatants were determined by ELISA and are expressed as means ± standard deviations for triplicate cultures. ∗∗, P < 0.01 versus the respective control. The results are representative of those from four different experiments.

FIG. 6

Induction of IL-8 release by p-TUA in THP-1 cells in culture. The OCT-differentiated THP-1 cells were stimulated by p-TUA and reference Salmonella serovar Abortus-equi LPS at the indicated concentrations for 24 h in triplicate. The IL-8 levels in THP-1 cell culture supernatants were determined by ELISA and are expressed as means ± standard deviations. ∗∗, P < 0.01 versus medium alone. The results are representative of those from three different experiments.

FIG. 7

Blocking effect of anti-human CD14 MAb on IL-8 secretion by THP-1 cells in response to p-TUA. The OCT-differentiated THP-1 cells were preincubated with anti-human CD14 MAb MY4 or isotype-matched control antibody (10 μg/ml) for 30 min and then stimulated with p-TUA (50 μg/ml) or reference LPS (10 ng/ml) for 24 h in triplicate. The IL-8 levels in the culture supernatants were determined by ELISA and are expressed as means ± standard deviations. ∗∗, P < 0.01 versus the respective control. The results are representative of those from three different experiments.

FIG. 8

Blocking effect of anti-human TLR4 MAb on IL-8 secretion by THP-1 cells in response to p-TUA. The OCT-differentiated THP-1 cells were preincubated with anti-human TLR4 MAb HTA125 or isotype-matched control antibody (5 μg/ml) for 30 min and then stimulated with p-TUA (50 μg/ml) or reference LPS (10 ng/ml) for 24 h in triplicate. The IL-8 levels in the culture supernatants were determined by ELISA and are expressed as means ± standard deviations. ∗∗, P < 0.01 versus the respective control. The results are representative of those from three different experiments.

FIG. 9

Inhibitory effect of LA-14-PP on IL-8 secretion by THP-1 cells in response to p-TUA. The OCT-differentiated THP-1 cells were preincubated with 1 or 10 μg of LA-14-PP per ml for 30 min and then stimulated with p-TUA (50 μg/ml) or reference LPS (10 ng/ml) for 24 h in triplicate. The IL-8 levels in the culture supernatants were determined by ELISA and are expressed as means ± standard deviations. ∗∗, P < 0.01 versus the respective control. The results are representative of those from three different experiments.

FIG. 10

Inability of p-TUA-[H] to induce cytokines in J774.1 and THP-1 cells in culture. J774.1 (A) and the OCT-differentiated THP-1 (B) cells were stimulated with p-TUA, p-TUA-[H], and reference LPS at the indicated concentrations for 24 h in triplicate. The TNF-α and IL-8 levels in the culture supernatants of J774.1 and THP-1 cells, respectively, were determinated by ELISA and are expressed as means ± standard deviations. ∗∗, P < 0.01; ∗, P < 0.05 (versus medium alone). The results are representative of those from three different experiments.